PROJECT SUMMARY Isoprenoids are a diverse, ubiquitous family of molecules with limitless industrial and clinical potential. As the largest family of secondary metabolites produced on Earth, isoprenoids also represent a fundamental building block for life. Consistent with this, organisms from all three domains of life synthesize isoprenoids that support critical metabolic and physiological processes. In bacteria, isoprenoids are necessary for the production of pigments, respiratory cofactors, and essential components of the cell envelope. Obstructing production of these molecules has devastating consequences for bacterial cells, supporting the idea that isoprenoid precursor synthesis enzymes are therapeutic targets for the treatment of pathogenic microorganisms. Although it has been established that numerous processes that rely on isoprenoids are required for virulence, the importance of isoprenoid production to bacterial pathogenesis has not been directly assessed. Additionally, the mechanism(s) by which bacteria synthesize and efficiently allocate isoprenoid precursors represent significant gaps in our knowledge. In bacteria isoprenoids are produced via a series of condensation and elongation reactions that use universal precursors as substrates to produce molecules of varying chain length. The reactions are catalyzed by enzymes referred to as prenyl diphosphate synthases (PDS). A growing body of evidence supports a model whereby short chain PDS and long chain PDS both synthesize the isoprenoid precursors used to produce the final products. However, long chain PDS are typically essential, making it difficult to test this model. In Staphylococcus aureus, the short chain and long chain PDS are encoded by ispA and hepT, respectively. Notably, S. aureus ispA mutants do not produce the isoprenoid-dependent pigment staphyloxanthin, however, production of the other isoprenoids appears to remain intact. Our preliminary data demonstrates that S. aureus hepT mutants are viable, enabling us to determine how this long chain PDS contributes to isoprenoid production. Additionally, we isolated staphyloxanthin-producing ispA suppressor mutants and mapped the mutations to hepT. These data reveal for the first time that hepT plays an important role in S. aureus isoprenoid production and allocation. We hypothesize that IspA and HepT function cooperatively to efficiently synthesize and allocate isoprenoid precursors to maintain maximal fitness during infection. To capitalize on our preliminary data and test our hypothesis, we will monitor the production of numerous isoprenoid-derived molecules in ispA and hepT mutants using established genetic, biochemical, and mass spectrometry approaches. A well-defined murine model of infection will quantify virulence of ispA and hepT mutants. Together these approaches will establish the contributions of IspA and HepT to S. aureus isoprenoid production during infection. Completion of this work will define the roles of IspA and HepT in an important human pathogen and validate the proteins as novel therapeutic targets.